PROJECT SUMMARY: Advances in high-throughput next generation sequencing (NGS) have enabled the rapid accumulation of pathogen-derived genomes for mining candidate virulence and antibiotic resistance genes, drug and vaccine targets, and many other genes of interest. These technologies also have facilitated studies of the microbiome and host susceptibility genetics. Advances in long-read sequencing contribute to higher quality genome assemblies and facilitate comparisons of closely related bacteria. RNA sequencing analyses complement whole genome sequencing data by providing additional insight into host-pathogen interactions, specifically a global view of the host cell responses to infections and regulation of pathogen-encoded virulence genes at multiple time-points during an infection. More recently, new approaches, such as single cell RNA sequencing, have enabled a more discrete examination of the cellular response to infection and the transduction of signals from an infected cell to adjacent cells. Applications of specialized molecular libraries (e.g. Hi-C and 10X Genomics) and minimal-infrastructure long-read DNA sequencing (e.g. Oxford Nanopore) show promise to produce high quality genome assemblies more effectively and efficiently, perhaps even from contaminated samples for which the target organism may be one of several bacteria present. In this GCID program, the Sequencing and Technology (ST) Core will leverage existing large-scale NGS infrastructure to evaluate the merits of new technologies and determine the most cost effective and expedient means to generate high quality genome assemblies for target pathogens. The experimental models developed in the four research projects (bacteria, virus, fungus, and parasite) will provide maximum utility to assess the contribution of these new technologies while simultaneously addressing questions related to infectious disease genomics using proven methods